This invention pertains generally to assay devices and methods for the detection of microscopic pathogens such as viruses or bacteria in a sample.
Methods for detecting the presence of biological substances and chemical compounds in samples has been an area of continuous development in the field of analytical chemistry and biochemistry. Various methods have been developed that allow for the detection of various target species in samples taken from sources such as the environment or a living organism. Detection of a target species is often necessary in clinical situations before an illness may be diagnosed and a prescribed method of treatment may be undertaken.
Several types of assay currently exist for detecting the presence of target species in samples. One conventional type of assay is the radioimmunoassay (RIA). RIA is a highly sensitive technique that can detect very low concentrations of antigen or antibody in a sample. RIA involves the competitive binding of radiolabeled antigen and unlabeled antigen to a high-affinity antibody. Typically, the labeled antigen is mixed with the antibody at a concentration that just saturates the antigen-binding sites of the antibody molecule. Then, increasing amounts of unlabeled antigen of unknown concentration are added. Because the antibody does not distinguish between labeled and unlabeled antigen, the two types of antigen compete for the available binding sites on the antibody. Measuring the amount of labeled antigen free in solutions, it is possible to determine the concentration of unlabeled antigen. Kuby, J., Immunology, W. H. Freeman and Company, New York, N.Y. (1991), pp. 147-150.
Another type of assay which has become increasingly popular for detecting the presence of pathogenic organisms is the enzyme-linked immunosorbent assay or ELISA. This type of assay allows pathogenic organisms to be detected using biological species capable of recognizing epitopes associated with proteins, viruses and bacteria. Generally, in an ELISA assay, an enzyme conjugated to an antibody will react with a colorless substrate to generate a colored reaction product if a target species is present in the sample. Kuby, J., Immunology, W. H. Freeman and Company, New York, N.Y. (1991), pp. 147-150. Physically adsorbed bovine serum albumin has been used in various such assays as a blocking layer because it has been found to prevent the non-specific adsorption of biological species that might interfere with or result in erroneous assay results.
Although ELISA and other immunosorbent assays are simple and widely used methods, they have several disadvantages. Tizard, I. R. Veterinary Immunology: An Introduction, W.B. Saunders Company, Philadelphia, Pa. (1996); Harlow, Ed.; Lane, D. Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y. (1988); Van Oss, C. J.; van Regenmortel, M. H. V. Immunochemistry, Dekker, New York, N.Y. (1994). Labeled antibodies can be expensive, especially for assays requiring radioactive labels. Additionally, radioactive labels require special handling as radioactive materials are also hazardous. The labeling of a compound, which is the main drawback of these methods, may alter the binding affinity of antibody to analyte. Enzymes are large molecules that may sterically inhibit antibody activity or it may lose enzymatic activity after conjugation to antibodies. Another concern with immunosorbent assays is non-specific binding of proteins to the solid support, antigen, and antibody complexes. This can lead to an increase in background noise, loss of sensitivity, and potentially a false positive test result. Additionally, the immobilization of proteins on the solid support can affect the conformation of the binding sites, leading to a decrease in sensitivity, and possible increase in non-specific binding. For example, physical adsorption of proteins to polystyrene wells occurs due to hydrophobic interactions between the protein and solid support. These interactions can also promote unfolding of the amino acid chains in order to cover the polystyrene surface. This can lead to possible inactivation of the binding sites.
Qualitative diagnostic assays based on aggregation of protein coated beads can also be used for the detection of proteins and viruses. Tizard, I. R. Veterinary Immunology: An Introduction, W.B. Saunders Company, Philadelphia, Pa. (1996): Cocchi, J. M.; Trabaud, M. A.; Grange, J.; Serres, P. F.; Desgranges, C. J. Immunological Meth., 160, (1993), pp. 1; Starkey, C. A.; Yen-Lieberman, B.; Proffitt, M. R. J. Clin. Microbiol., 28, (1990), pp. 819; Van Oss, C. J.; van Regenmortel, M. H. V. Immunochemistry, Dekker, New York, N.Y. (1994). For direct detection of antibodies, antigen is non-specifically adsorbed to the surface of latex beads which are several microns in diameter. The protein-coated beads possess a slight charge which prevents aggregation. Introduction of an antibody specific to the adsorbed protein can link the beads, leading to agglutination. The agglutination can be detected by eye or by other methods such as quasi-elastic light scattering. Visual agglutination assays, however, are not sensitive and measurement by quasi-elastic light scattering requires complex apparatus and is not suitable for use in locations remote from central labs. Furthermore, it is not possible to perform highly multiplexed agglutination assays using microarrays because of the bulk solution methodology of this type of assay.
To overcome the need for labeled proteins, principles based on direct detection of the binding of proteins and ligands have been investigated. Schmitt, F.-J.; Haussling, L.; Ringsdorf, H.; Knoll, W. Thin Solid Films, 210/211, (1992), pp. 815; Hauslling, L.; Ringsdorf, H. Langmuir, 7, (1991), pp. 1837. Surface plasmon reflectometry (SPR) is one such method. SPR is sensitive to changes in the index of refraction of a fluid near a thin metal surface that has been excited by evanescent electromagnetic waves. The binding of proteins to ligands can be detected by examining an increase in the resonance angle or intensity of signal. Typical angular resolution using this method is 0.005xc2x0 allowing detection of sub-angstrom changes in adsorbed film thickness with SPR. However, care must be taken to ensure that the change in resonance angle is due to binding and not just a change in the bulk solution index of refraction. A thermally stable environment is required due to the dependence of the resonance angle on the index of refraction of the fluid. An increase in temperature from 25xc2x0 C. to 26xc2x0 C. in water amounts to a change in the index of refraction by 0.0001. This increase would result in the change in resonance angle of approximately 0.015xc2x0 or roughly 0.2 nm in the observed height of a protein layer. This temperature stability requirement makes SPR unsuitable for most field applications. In addition, non-specific adsorption of molecules on to or near the sensor surface can lead to false changes in signal, requiring a surface which minimizes non-specific interactions. Therefore, surface plasmon reflectivity is more complex than ELISA, requires laboratory based equipment, and the preparation of a well defined surface.
The use of ion-channel switches for detecting biospecific interactions has been reported. Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature, 387, (1997), pp. 580. In a device using ion channel switches, a tethered lipid membrane incorporating mobile ion channels is separated from a gold electrode surface by an ion reservoir. The gold surface serves as an anchor for the membrane and acts as an electrode. Within the membrane are upper and lower ion channels. In order to become conductive, the outer and inner ion channels must align and form a dimer. Membrane spanning lipids, which help stabilize the lipid membrane, are attached at one end to the electrode surface and are terminated with ligands that extend away from the membrane. The ion channels of the outer layer possess ligands. Unbound, the outer ion channels move freely, occasionally forming dimers with the inner channels, allowing conduction. The binding of a bivalent molecule to both the ion channel and membrane spanning lipid restricts the mobility of the outer ion channel, leading to a measurable decrease in conductivity. However, if a large amount of protein adsorbs to the outer layer, the ion channel mobility presumably would be restricted and a false decrease in conductance could be observed due to non-specific interactions. Additionally, this method requires sensitive devices for detecting the change in conductance. The procedure for fabricating the membranes requires several hours and the membrane stability is limited (must be immersed in solution). More importantly, specific antibodies must be attached to the membrane/channels, requiring separate protein chemistry for each analyte to be detected.
A method based on a porous silicon support that permits optical detection of the binding of specific proteins to ligands has been reported. Lin, V.; Motesharei, K.; Dancil, K. S.; Sailor, M. J.; Ghadiri, M. R. Science, 278, (1997), pp. 840; Dancil, K. S.; Greiner, D. P.; Sailor M. J. J. Am. Chem. Soc., 121, (1999), pp. 7925. The porous areas are typically 1 to 5 xcexcm deep and a few square micrometers to millimeters in area. Typical binding times are on the order of 30 minutes followed by rinsing of the surface. Initial work in this area incorrectly reported the detection of extremely low concentrations of analyte. Binding of streptavidin to biotinylated surfaces was initially found to reduce the index of refraction of the porous support, however this was later correctly attributed to an oxidation of the surface. In addition, a change in the effective optical thickness of the film was reportedly observed upon introduction of streptavidin, however they could not differentiate between specific interactions and non-specific adsorption. This method does not require labeled molecules, however, the porous silicon surface is susceptible to oxidation and non-specific adsorption.
The use of polymerized multilayer assemblies for the detection of receptor-ligand interactions has also been reported. Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science, 261, (1993), pp. 585; Pan, J. J.; Charych, D. Langmuir, 13, (1997), pp. 1365. Polydiacetylene multilayer films deposited by Langmuir-Blodgett technique change color from blue to red due to a conformational change in the polymer backbone. For example, changes in temperature or pH can cause a shift in color. The response can be controlled and used for protein detection by attaching ligands to the multilayer. Upon binding of a multivalent macromolecule to ligands, stress is introduced into the multilayer assembly. A change in color is seen in the system if sufficient protein is bound, with binding times typically on the order of 30 minutes. This system permits direct detection of receptor-ligand interactions and transduces the events into an optical signal that can be easily measured and quantified. The optical output can be interpreted by eye or analyzed with a spectrophotometer for quantitative conclusions. The use of polymerized multilayer assemblies for the detection of influenza virus has been demonstrated. A significant disadvantage of this method, however, is that it requires multi-valent analyte. Multiple ligands connected to the polymerized multilayer must attach to the same macromolecule. This prevents the use of this method for monovalent molecules (even bead based assays can be performed competitively, not requiring multivalent molecules). Binding of bivalent molecules such as IgG""s has not been demonstrated. Furthermore, Langmuir-Blodgett deposition is a process which is difficult to translate from laboratory to commercial scale. As an alternative method to Langmuir-Blodgett deposition, these principles has also been demonstrated using vesicles. However, research based on vesicles, reveals the usefulness of the system to be limited because it is insensitive to the analyte at concentrations below 0.1 mg/ml.
Although many of the conventional assay methods described above work very well to detect the presence of target species, many conventional assay methods are expensive and often require instrumentation and highly trained individuals, which makes them difficult to use routinely in the field. Thus, a need exists for assay devices and systems which are easier to use and which allow for evaluation of samples in remote locations.
Recently, assay devices that employ liquid crystals have been disclosed. For example, a liquid crystal assay device using mixed self-assembled monolayers (SAMs) containing octanethiol and biotin supported on an anisotropic gold film obliquely deposited on glass has recently been reported. Gupta, V. K.; Skaife, J. J.; Dubrovsky, T. B., Abbott N. L. Science, 279, Mar. 27, 1998, pp. 2077-2079. In addition, PCT publication WO 99/63329 published on Dec. 9, 1999 discloses assay devices using SAMs attached to a substrate and liquid crystal layer which is anchored by the SAM. Although the disclosed liquid crystal-based assay devices which use anisotropic gold films are suitable for use in determining whether a target protein is present in a sample, the preparation of the anisotropic gold film by oblique deposition is difficult. For example, the preparation of the obliquely deposited gold films requires complicated cleaning steps and high vacuum deposition. Further, such assay devices are suited to the detection of protein molecules in a sample rather than larger complex particles such as viruses and other pathogens.
In accordance with the present invention, microscopic pathogens such as bacteria and viruses may be detected in a simple and efficient manner. The detection of a pathogen can be carried out by personnel who have minimal training and without requiring specialized laboratory facilities or equipment. Detection is provided with accurate readout in a manner that is faster than conventional serological tests. It is possible to screen for multiple microscopic pathogens in a single test.
A detection apparatus in accordance with the invention includes a substrate having a detection region thereon comprising microstructures which include depressions of width and depth sized to align liquid crystal material in contact therewith. The depressions are also sized to be occupied by the pathogen to be detected. The depressions may comprise parallel microgrooves. For detection of viruses, the width and depth of the microgrooves will generally be on the order of 5 nm to 500 nm, while for detection of bacteria the width and depth of the microgrooves will generally be on the order of 0.1 xcexcm to 10 xcexcm. The microstructure on the substrate may be formed by various suitable technologies, including molding a hardenable polymer material, utilizing a micromachined mold, and by other methods such as mechanical embossing or by using an elastomeric material such as polydimethylsiloxane to form a replica from a master and then using the elastomeric replica to form replicas from other polymeric materials including polyurethane, polycyanoacrylate, and polystyrene. The surface of the detection region is treated to block nonspecific binding of pathogens to the surface and includes a binding agent that specifically binds the selected pathogen to be detected.
The detection apparatus may be further embodied in that at least a portion of the detection region may be coated with an inorganic material such as an oxide of silicon, an oxide of a metal, a metal, or combinations of these. A metal coated region such as a gold or silver coated region may include a region that is the reaction product of the metal coated region with a mercaptan or a disulfide. In still other embodiments, substantially all the binding agent is located in the depressions on the surface of the detection region.
To test a sample for the presence of the selected pathogen, the sample is contacted to the surface of the detection region to permit the pathogen particles, if present, to be bound to the surface by the binding agents and to occupy the depressions. A liquid crystal material is thereafter applied to the detection region that will be aligned by the microstructures on the surface of the substrate in the absence of binding of particles to the surface. Where no particles are present on the surface, the liquid crystal material is aligned and appears uniform and dark when visually examined, typically by utilizing an appropriate polarizing viewing material. If the specific pathogen is present in the sample, it will be bound to the binding agents and will substantially occupy the depressions on the surface of the substrate. The occupation of the depressions by the particles disrupts the uniform alignment of the liquid crystal material, with the result that the detection region appears relatively brightly colored when viewed with the appropriate polarizing material. In this manner, an observer can readily and easily determine whether or not the specific pathogen is present in the sample. The depressions on the substrate are sufficiently large compared to molecular material such as proteins which may be bound to the surface, and thus the non-specific binding of proteins or other molecular materials does not disrupt the uniform alignment of the liquid crystal material.
Multiple substrates can be used or the surface of the detection region of a single substrate may be patterned such that the various areas of the detection region each have a binding agent for a different specific pathogen. This allows a single sample to be simultaneously checked for the presence of a variety of pathogens. The detection apparatus may further be embodied in a microarray in which multiple detection regions are provided on a unitary substrate to facilitate the screening of a sample for the presence of several pathogens. Additionally, the detection apparatus may be further embodied in that it contains two or more detection regions such that the detection regions have grooves of different widths, depths or both such that the different regions of the detection apparatus have different sensitivities to a single specific pathogen.
An exemplary material that may be used to block nonspecific absorption of viruses is bovine serum albumin (BSA). An exemplary binding agent is an immunoglobulin or portion thereof or an antibody or portion thereof selected for binding to a specific virus. However, it is understood that any suitable blocking layer material and binding agent may be utilized in the present invention.
In further embodiments of the invention, magnetic beads may be used in conjunction with the detection apparatus and methods of the present invention. In such embodiments, the surface of the beads may include a binding agent that binds a selected pathogen. The beads may be contacted with a sample and subsequently contacted with the detection region. If the pathogen is present in the sample, it will bind to the surface of the beads such that the bead binds to the binding agent in the detection region of the detection apparatus.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.